Received: 11 July 2023 Accepted: 20 October 2023

DOI: 10.1111/1462-2920.16532

RESEARCH ARTICLE

Complex interactions between diverse mobile genetic

elements drive the evolution of metal-resistant bacterial
genomes

Khandaker Rayhan Mahbub 1 | Caroline Chénard 2 | Steven Batinovic 3 |

Steve Petrovski 4,5 | Federico M. Lauro 2,6,7 | Md Hafizur Rahman 1 |
Mallavarapu Megharaj 8,9 | Ashley E. Franks 4,5 | Maurizio Labbate 1

1
School of Life Sciences, University of
Technology Sydney, Ultimo, New South
Wales, Australia
2
Asian School for the Environment, Nanyang
Technological University, Singapore,
Singapore
3
Division of Materials Science and Chemical
Engineering, Yokohama National University,
Yokohama, Kanagawa, Japan
4
Department of Physiology, Anatomy and
Microbiology, La Trobe University, Bundoora,
Victoria, Australia
5
Centre for Future Landscapes, La Trobe
University, Bundoora, Victoria, Australia
6
Singapore Centre for Environmental Life
Sciences Engineering/Nanyang Technological
University, Singapore, Singapore
7
Nanyang Environment & Water Research
Institute (NEWRI), Nanyang Technological
University, Singapore, Singapore
8
Global Centre for Environmental
Remediation, College of Engineering, Science
and Environment, The University of
Newcastle, Callaghan, Australia
9
Cooperative Research Centre for
Contamination Assessment and Remediation
of Environment, The University of Newcastle
(UoN), Callaghan, New South Wales,
Australia
Correspondence
Maurizio Labbate, School of Life Sciences,
University of Technology Sydney, NSW 2007,
Australia.
Email: maurizio.labbate@gmail.com
Khandaker Rayhan Mahbub, South Australian
Research and Development Institute, Primary
Industries and Regions SA, Urrbrae, SA 5064,
Australia.
Email: krmjissan@gmail.com; khandaker.
mahbub@sa.gov.au
Abstract
In this study, we compared the genomes of three metal-resistant bacteria
isolated from mercury-contaminated soil. We identified diverse and novel
MGEs with evidence of multiple LGT events shaping their genomic structure
and heavy metal resistance. Among the three metal-resistant strains, Sphin-
gobium sp SA2 and Sphingopyxis sp SE2 were resistant to multiple metals
including mercury, cadmium, copper, zinc and lead. Pseudoxanthomonas
sp SE1 showed resistance to mercury only. Whole genome sequencing by
Illumina and Oxford Nanopore technologies was undertaken to obtain
comprehensive genomic data. The Sphingobium and Sphingopyxis strains
contained multiple chromosomes and plasmids, whereas the Pseudox-
anthomonas strain contained one circular chromosome. Consistent with
their metal resistance profiles, the strains of Sphingobium and Sphingopyxis
contained a higher quantity of diverse metal resistance genes across their
chromosomes and plasmids compared to the single-metal resistant Pseu-
doxanthomonas SE1. In all three strains, metal resistance genes were prin-
cipally associated with various novel MGEs including genomic islands (GIs),
integrative conjugative elements (ICEs), transposons, insertion sequences
(IS), recombinase in trio (RIT) elements and group II introns, indicating their
importance in facilitating metal resistance adaptation in a contaminated
environment. In the Pseudoxanthomonas strain, metal resistance regions
were largely situated on a GI. The chromosomes of the strains of Sphingo-
bium and Sphingopyxis contained multiple metal resistance regions, which
were likely acquired by several GIs, ICEs, numerous IS elements, several
Tn3 family transposons and RIT elements. Two of the plasmids of Sphingo-
bium were impacted by Tn3 family transposons and ISs likely integrating
metal resistance genes. The two plasmids of Sphingopyxis harboured trans-
posons, IS elements, an RIT element and a group II intron. This study pro-
vides a comprehensive annotation of complex genomic regions of metal
resistance associated with novel MGEs. It highlights the critical importance
of LGT in the evolution of metal resistance of bacteria in contaminated
environments.

Present addresses
Khandaker Rayhan Mahbub, South Australian
Research and Development Institute, Primary
Industries and Regions SA, Urrbrae, South

© 2023 Applied Microbiology International and John Wiley Sons & Ltd.

Environ Microbiol. 2023;1–19. wileyonlinelibrary.com/journal/emi 1


2 MAHBUB ET AL.
Australia, Australia; Caroline Chénard, Aquatic
and Crop Resource Development-National
Research Council Canada, Halifax, Nova
Scotia, Canada; and Maurizio Labbate,
AusDiagnostics Pty Ltd, Mascot, New South
Wales, Australia.
INTRODUCTION
Soils are important for fundamental natural biogeo-
chemical cycles, detoxification of organic and inorganic
pollutants, and provision of nutrition for plants that feed
into the animal and human food chain (Lorenz &
Lal, 2009; Smith et al., 2015); however, these funda-
mental functions are threatened due to metal pollution
(Wu et al., 2016). Mercury (Hg), cadmium (Cd), lead
(Pb), chromium (Cr) and arsenic (As) do not have any
known biological functions and are toxic, while metals
such as cobalt (Co), zinc (Zn), nickel (Ni) and copper
(Cu) serve as cofactors for biological reactions but can
be toxic at certain concentrations (Kumar et al., 2017;
Liu et al., 2018). In soils, metals undergo various chem-
ical reactions, accumulate in biotic tissues, biologically
augment the food web and exert acute to chronic toxic-
ity on plant, animal and human health (Mahbub
et al., 2018; Skyllberg, 2012). Additionally, polluting
metals have profound impacts on the structure of
microbial soil communities (Mahbub et al., 2020; Zhao
et al., 2020). Bacteria have evolved diverse mecha-
nisms to control the uptake, efflux or detoxification of
metals. The three basic mechanisms for bacterial resis-
tance to toxic metals include—(a) detoxification or bio-
transformation into a non-toxic or less toxic form,
(b) extrusion of the metals from the cell via efflux
pumps and (c) compartmentalization of the metals
within or outside the cells (Hao et al., 2021; Rahman &
Singh, 2020). Genes encoding these resistance mech-
anisms are diverse and are essential in bioremediation
strains including those that might be designed by syn-
thetic biology techniques (Mahbub et al., 2017; Padhan
et al., 2021).
Many mobile genetic elements (MGEs) carrying
metal resistance genes (MRGs) have been character-
ized, demonstrating lateral gene transfer (LGT) as a
driver in bacterial adaptation to metal pollutants
(Brito, 2021; Hemme et al., 2016; Larbig et al., 2002; Li
et al., 2018). LGT is a two-step process requiring
(i) physical movement of DNA between cells
(e.g., transformation) and (ii) successful integration of
the transferred DNA into the chromosome or a resident
plasmid(s) (Soucy et al., 2015). Plasmids being self-
replicating do not require integration. While integration
of laterally acquired DNA may occur through homolo-
gous recombination, MGEs such as insertion
sequences (ISs) and transposons, genomic islands
(GIs) and integrative conjugative elements (ICEs) are
evolutionarily advantageous because they are able to
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accumulate diverse genes and insert in genomes with-
out the requirement for DNA homology (Frost
et al., 2005). GIs and ICEs are long mobile DNA seg-
ments (approximately 10–200 Kb) with features such
as differing GC content, located near tRNA sites and
being flanked by inverted repeats (IRs) and/or, direct
repeats (DRs) that are generated during insertion
(Delavat et al., 2017; Juhas et al., 2009). Unlike GIs,
ICEs encode conjugative genes that facilitate conjuga-
tive transfer of the element to other cells (Delavat
et al., 2017). Excision and integration of GIs and ICEs
occur at defined sites and are catalysed by an
integrase(s) encoded on the element itself. An emerg-
ing integrative MGE relevant to this study is recombi-
nase in trio (RIT) elements consisting of three adjacent
tyrosine-based site-specific recombinases (Ricker
et al., 2013).
ISs and transposons are segments of DNA that
(Hickman et al., 2015) are regularly found in multiple
copies in the genome of the same cell. Traditionally,
characteristics such as size and the presence of pas-
senger genes define the difference between IS ele-
ments and transposons although this distinction is
blurred (Partridge Sally et al., 2018). Most IS elements
and transposons contain IRs that are recognized by a
transposase(s) (e.g., TnpA) encoded on the element,
which facilitates excision and insertion into a new site
and, often creating short flanking DRs of 3–14 bp
(Siguier et al., 2015). For some transposons and ISs,
other genes may be involved depending on their mode
of transposition. Some IS elements such as those of
the IS91 and ISCR families use transposition mecha-
nisms that sometimes move adjacent regions (Siguier
et al., 2015). IS copies create further genetic diversity
by homologously recombining resulting in DNA inver-
sions (if ISs are inverted relative to one another) or,
DNA excision forming a circle containing the interven-
ing DNA and a single copy of the IS (if ISs are in the
same orientation). Excised DNA circles may relocate
into a new genetic site via homologous recombination
such as with another IS. Additionally, if present in tan-
dem, ISs can move together as a single unit called a
composite transposon carrying the DNA between them
(Siguier et al., 2015). ISs and transposons may acquire
passenger genes such as transposons of the Tn3 fam-
ily, which often contain Hg resistance genes (Nicolas
et al., 2015; Siguier et al., 2015). Tn3 family transpo-
sons contain a transposase (tnpA) and a resolvase
(tnpR) and use a paste-and-copy replicative mode of
transposition generating a cointegrate intermediate

COMPLEX GENOMIC REGIONS OF METAL RESISTANCE ASSOCIATED WITH NOVEL MGES
between donor and target molecules that is resolved by
the resolvase (Nicolas et al., 2015).
Although MGEs linked to MRGs have been charac-
terized, how bacteria and their genomes adapt to soils
heavily contaminated with multiple metals is less under-
stood. To address this, we used short-read Illumina
and long-read Oxford Nanopore sequencing to carefully
interrogate the genomes of three metal-resistant bacte-
rial strains namely, Sphingobium sp. SA2, Sphingo-
pyxis sp. SE2 and Pseudoxanthomonas sp. SE1.
Sphingobium and Sphingopyxis belong to a group of
Gram-negative, aerobic and chemoheterotrophic bacte-
ria with sphingolipid-rich membranes that are com-
monly isolated from soil and water environments
(Takeuchi et al., 2001). This group of bacteria is known
for their resistance to various organic pollutants and
degradation of xenobiotic and organic compounds (Fu
et al., 2014; Kuroda et al., 2017; Li et al., 2013; Zhao
et al., 2017); however, their ability to resist and detoxify
metals is not well understood. There are only a few
studies where different strains from this group are
reported to tolerate metals such as Ni, Pb, Cd, Cu and
Zn (Chen et al., 2016; D’Argenio et al., 2014; Li
et al., 2013; Wang et al., 2013), including our reports
on Hg resistance of the strains Sphingobium sp. SA2
and Sphingopyxis sp. SE1, of which Sphingobium
sp. SA2 was able to rapidly volatilize Hg from soil
microcosms (Mahbub, Krishnan, Megharaj, &
Naidu, 2016). Pseudoxanthomonas sp. SE1 is a Gram-
negative pollutant degrader and is Hg-resistant
(Mahbub, Krishnan, Naidu, & Megharaj, 2016). Our
analyses show the genomes of these strains to be
heavily impacted by MGEs including plasmids, GIs,
ICEs, ISs, transposons and RITs. We identify novel
metal-resistant MGEs and comprehensively describe
the metal-resistance genetic loci of these strains show-
ing that the evolution of metal resistance is largely
driven by complex interactions of different MGEs.
EXPERIMENTAL PROCEDURES
Strains and culture conditions
The experimental strains Sphingobium sp. SA2, Sphin-
gopyxis sp. SE2 and Pseudoxanthomonas sp. SE1 were
isolated previously from Hg-contaminated soil in Botany
Bay, Sydney, Australia (Mahbub et al., 2017; Mahbub,
Krishnan, Megharaj, & Naidu, 2016; Mahbub, Krishnan,
Naidu, & Megharaj, 2016). The strains were cultured
from frozen stocks on LB agar (1% tryptone, 0.5% yeast
extract, 1% NaCl and 1.5% agar) at 30� C for 3–5 days.
For routine microbiological analyses, LB broth was uti-
lized. For metal resistance assays, a low phosphate
medium (LP broth) was prepared as follows: 1.95 g/L
2-(N-morpholino)ethanesulfonic acid; 0.01 g/L Na2HPO4;
0.05 g/L NH4Cl; 0.02 g/L KCl; 0.24 g/L MgSO4.7H2O;
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3
0.14 g/L CaSO4; 0.004 g/L FeSO4.7H2O. The medium
was supplemented with 0.1% SL7 trace solution (0.1%
HCl, 35 mg/L ZnCl2, 50 mg/L MnCl2.H2O, 30 mg/L
H3BO3, 200 mg CoCl2.2H2O, 20 mg/L CuCl2.2H2O,
20 mg/L NiCl2.6H2O, 40 mg/L NaMoO4.2H2O) and 0.2%
Glucose. The pH was adjusted to 6.4 by 10 N NaOH.
Divalent heavy metal resistance assay
Four heavy metals namely Cd, Pb, Zn and Cu were
selected to determine the multi-metal resistance pattern
of the three strains Sphingobium sp. SA2, Sphingo-
pyxis sp. SE2 and Pseudoxanthomonas sp. SE1. Low
phosphate-containing LP medium was used as it pro-
vides higher availability of the added heavy metals to
the bacterial strains than complex nutrient media
(Mahbub, Krishnan, Naidu, & Megharaj, 2016;
Rathnayake et al., 2013). The medium was autoclaved
and supplemented with different concentrations of filter-
sterilized heavy metals such as CdCl2, Pb(NO3)2, ZnCl2
and CuCl2. Based on preliminary observation of the
strains’ metal-tolerance ranges, experimental doses of
the tested metals were determined as follows: Cd—0,
5, 7, 10, 15, 20, 30 and 40 mg/L; Pb—0, 10, 20,
40, 60 and 80 mg/L; Cu—0, 1, 2, 3, 4, 5, 6 and 7 mg/L
and Zn—0, 10, 20, 40, 60, 80, 100 and 150 mg/L.
Media without metal supplementation were used as
controls. Controls and metal-supplemented LP media
were dispensed into 48-well microtitre plates and inocu-
lated with 10% (v/v) overnight grown experimental cul-
ture to a final optical density (OD) of 0.09 and 600 nm.
Microtitre plates were then closed, sealed with parafilm
and incubated at 25� C in the dark. Bacterial growth
was measured through monitoring of OD at 600 nm
after 72 h using the Bio-Tek Synergy HT Multi-
Detection Microplate Reader equipped with KC4 soft-
ware. All concentrations and metals were examined in
triplicate. The EC50 values are the statistically derived
estimated concentration that produces a 50% reduction
of bacterial growth after 72 h and was determined by
applying a non-linear regression model with a four-
parameter logistic curve utilizing IBM SPSS 17 Statisti-
cal Software.
Genomic DNA extraction and DNA
sequencing
DNA was extracted using the Isolate II Genomic DNA
Kit (Bioline). Illumina sequence data were obtained by
generating Nextera libraries followed by Illumina MiSeq
sequencing resulting in read depths of 108-fold, 59-fold
and 72-fold generated for SE1, SE2 and SA2, respec-
tively. For Oxford Nanopore Technologies (ONT) Mini-
ION sequencing, libraries were prepared from 1.5 μg
gDNA using the ONT 1D ligation sequencing kit

4 MAHBUB ET AL.
(SQK-LSK108) with the native barcoding expansion
kit (EXP-NBD103). Libraries were carefully prepared to
avoid shearing and maximize sequence read lengths.
AMPure XP beads were used to clean the DNA
between steps.
A 50 fmol equimolar library was loaded on a single
MinION SpotON flowcell, R9.4.1 (Oxford Nanopore)
MK1b device using the NC-48h_Sequencing_Run-
FLO-MIN106_SQK-LSK108 protocol for 48 h.
During and after the sequencing run, the fast5 read
files were base-called during and after the run using
ONT’s Albacore (v2.3.4). Albacore was run with barcode
demultiplexing and fastq output that removes
barcodes and adaptor sequences and, demultiplexes the
sequences. Read depths of 39-fold, 8-fold and 87-fold
were generated for SE1, SE2 and SA2, respectively.
Bioinformatics analyses
Illumina and ONT sequence reads were assembled
using Unicycler (v0.4.4) (Wick et al., 2017).
Assembled genomes were annotated using Prokka
1.12 (Seemann, 2014) and visualized for further ana-
lyses in Geneious Prime 2.2 (Kearse et al., 2012). Fur-
ther annotation of MRGs was carried out using the
BacMet database (Pal et al., 2013) and the NCBI data-
base (Johnson et al., 2008). Phylosift (Darling
et al., 2014) was used to identify the most closely
related closed genomes in the database for alignments
with MAUVE (Darling et al., 2004). GIs were identified
using a combination of IslandViewer 4 (Bertelli
et al., 2017) and alignments to closely related genomes
using MAUVE. A metal resistance region was defined
as more than one gene responsible for detoxification,
efflux or sequestration of metals clustered together on
a replicon/contig but not separated by more than 5 Kb.
Regulatory genes were not included in analyses. The
genetic context of solitary MRGs was not investigated,
however, were included in a gene copy analysis.
Where an MGE was identified overlapping a metal
resistance region, it was reclassified as a separate
region. IS elements were searched against the ISfinder
database and if novel, submitted to the database and
given a new designation (Siguier et al., 2006). Genetic
schematic representations were drawn by hand in
Microsoft PowerPoint.
RESULTS
Divalent heavy metal resistance profiles of
three Hg-bioremediation strains
The Hg resistant and bioremediation strains namely,
Sphingobium sp. SA2, Pseudoxanthomonas sp. SE1
and Sphingopyxis sp. SE2 were previously isolated
from Hg-contaminated soil and shown to volatilize Hg
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when added to in vitro cultures (Mahbub et al., 2017;
Mahbub, Krishnan, Megharaj, & Naidu, 2016). Among
the three strains, Sphingobium sp. SA2 was employed
to remove Hg from contaminated soils (having 100–
280 mg/kg Hg) with the strain removing 30%–60% of
soil-bound Hg from four soils with different properties
(Mahbub, Krishnan, Megharaj, & Naidu, 2016). As
many anthropogenically-impacted environments con-
tain multiple pollutants, particularly other heavy metals,
efficient Hg bioremediation strains should ideally be
capable of tolerating the presence of these other diva-
lent heavy metal pollutants (Alloway, 2013; Roberts
et al., 2005). To verify this, the three bacterial strains
were subjected to growth with different concentrations
of Cd, Pb, Cu and Zn. The toxicity tests were carried
out in a chemically defined minimal medium containing
less phosphate and organic substances because their
presence might chelate divalent metals and result in an
overestimation of toxicity data (Kumar et al., 2013;
Rathnayake et al., 2013).
Among the three Hg-resistant strains, SE1 did not
exhibit resistance to the tested heavy metals at the con-
centrations used showing no visible growth for the low-
est experimental concentrations of 1 mg/L for Cd, Pb,
Cu and Zn. Strains SA2 and SE2 showed resistance to
all four divalent metals (Figure 1). A sigmoidal growth
pattern was observed with a rapid decrease in
growth when concentrations reached toxic levels.
Based on the metal resistance profile (Figure 1), both
SA2 and SE2 are multi-metal resistant with some minor
differences. While both SA2 and SE2 have similar
resistance to Pb, SA2 had a 2.70-fold higher resistance
to Cd and 20-fold higher resistance to Zn compared
to SE2. On the other hand, Cu resistance was 2.23-fold
higher in SE2 compared to SA2.
Genome assemblies
To determine the genetic basis of metal resistance in
the multi-metal-resistant Sphingobium sp. SA2 and
Sphingopyxis sp. SE2 strains as compared to the sin-
gle metal Hg-resistant Pseudoxanthomonas sp. SE1
strain, genome sequencing was undertaken. Metal
resistance regions are often associated with repeat ele-
ments that complicate assembly therefore, a combina-
tion of Illumina and Oxford Nanopore sequencing was
undertaken to maximize producing closed genomes or
draft genomes with a low number of contigs. A sche-
matic representation of the genomes is presented in
Figure 2.
SE1 produced one closed circular chromosome of
4.23 Mb. The size of the SE1 genome is slightly higher
than the other closely related Pseudoxanthomonas
genomes in the database with sizes ranging from 3.42
to 3.89 Mb. Annotation with NCBI Prokaryotic Genome
Annotation Pipeline (PGAP) identified a total of 3889
protein-coding sequences (CDS), 6 ribosomal RNA
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COMPLEX GENOMIC REGIONS OF METAL RESISTANCE ASSOCIATED WITH NOVEL MGES 5

F I G U R E 1 Resistance profiles to the divalent heavy metals Cd2+, Pb2+, Cu2+ and Zn2+ for the Hg-bioremediation strains Sphingobium
sp. SA2, Sphingopyxis sp. SE2 and Pseudoxanthomonas sp. SE1. Upper panel shows toxicity patterns of SA2 and SE2. Data were plotted as
means of triplicate observations of optical density at 600 nm for the tested metal concentrations with error bars showing the standard deviations
of the means. Growth curves of strain Pseudoxanthomonas sp. SE1 could not be generated as it did not grow in the lowest experimental
concentrations. Table (below) shows the estimated EC50 values extrapolated from the fitted lines in the figures in the upper panel using the
fourth parametric logistic equation.

(rRNA), 50 transfer RNA (tRNA) and 1 transfer-
messenger RNA (tmRNA).
SA2 produced 5 circular contigs of 3.3 Mb, 1.2 Mb,
172 Kb, 66 Kb and 58 Kb with coverages of 1.00�,
1.41�, 1.72�, 6.42�, and 2.00�, respectively, and
1 uncircularised contig of 1.7 Kb. As a result, SA2 con-
tains one large chromosome of 3.3 Mb (CH1), a smaller
chromosome of 1.2 Mb (CH2) and three plasmids of
172 Kb (p172), 66 Kb (p66) and 58 Kb (p58). The multi-
ple chromosomes and plasmids in strain SA2 are con-
sistent with many other Sphingobium genomes in the
NCBI database. The size of the genome is consistent
with the existing sizes of the reported Sphingobium
genome that range from 4.08 to 5.9 Mb (Verma
et al., 2014; Zhao et al., 2017). PGAP identified a total
of 4689 CDS, 9 rRNA, 53 tRNA and 1 tmRNA in the
entire genome.
Assembly of the SE2 genome was more complex
as Unicycler produced just one circular contig of
231 Kb (contig 3) and 10 uncircularised contigs of vari-
ous lengths. Contigs 10 and 11 are smaller than 1 Kb
with Contig 10 identical to ISSph13 and possibly repre-
senting an excised IS element and Contig 11 not giving
any hit to known mobile elements. Consistent with
being a plasmid, contig 3 had a coverage of 2.73x. A
similar coverage of 2.65x was observed for contig
2 (434 Kb) and combined with the lack of
chromosomal-like genes and the presence of conjuga-
tive genes (e.g., traD), is presumed to be a plasmid.
Contigs 1 (4.3 Mb), 4 (61 Kb) and 5 (51 Kb) are hypoth-
esized to make up a single chromosome and are sup-
ported by their alignment to the single chromosome of
the closed genome of Sphingopyxis alaskensis
PAMC25046 and the presence of chromosomal-like
genes such as tRNA, 16 s rRNA and DNA polymerase
I and III. Contigs 6 (17 Kb), 7 (5 Kb), 8 (3 Kb) and
9 (2 Kb) did not align with the PAMC25046 genome.
The total size of the assembled chromosome of SE2 is
4.89 Mb and is within the range of other Sphingopyxis
strains (3.35–5.9 Mb). PGAP identified 4880 CDS,
3 rRNA, 46 tRNA and 1 tmRNA.
Genome sequencing reveals the presence
of multiple heavy MRGs
We characterized the presence of known MRGs and
their copy numbers. To assist the reader, a summary
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6 MAHBUB ET AL.

F I G U R E 2 Schematic representation of the Sphingobium sp. SA2, Sphingopyxis sp. SE2 and Pseudoxanthomonas sp. SE1 assembled
genomes and the relative position of metal resistance regions. Metal resistance regions are shown as red rectangles on the replicon and labelled
as the regions described in Table 1. In Sphingobium sp. SA2, p66 is the only replicon without metal resistance genes. In Sphingopyxis sp. SE2,
chromosome 1 (CH1) is predicted to consist of contigs 1, 4 and 5 based on their alignment to the single chromosome of the closed genome of
Sphingopyxis alaskensis PAMC25046. Contigs 6 (17 Kb), 7 (5 Kb), 8 (3 Kb) and 9 (2 kB) are not shown as their placement is unclear. Contig
2 did not produce a circularized sequence, however, is predicted to be a plasmid and is shown as circular in this image.

of MRGs described in this results section is provided
in Table S1. The genomes of SA2 and SE2 contained
genes encoding resistance to Hg, Cd, Zn, Co, Cu, As,
(Silver) Ag and Ni. Given the multi-metal-resistant
nature of SA2 and SE2, the frequency of MRGs in
these two strains is comparable (101 copies of MRGs
in SA2 and 102 copies of MRGs in SE2) (Figure 3).
In comparison, SE1 harboured fewer MRGs (47 copies
of MRGs) and lacked a number of MRGs present in
the other two genomes. In both SA2 and SE2, MRGs
are located across multiple replicons including both
chromosomes and two plasmids (p172 and p58) and,
across one chromosome, one plasmid (p231) and one
putative plasmid (contig 2), respectively (Table 1;
Figure 2). SE1 (Figure 3) contains genes encoding
resistance to Hg, Cd, Zn, Co, Cu, As and Ag
(Table 1) with most of these genes located across a
120 Kb region in its single chromosome (Table 1;
Figure 2).
Metal resistance regions show genetic
signatures of acquisition by LGT
Genetically linked MRGs were categorized into regions
(see section ‘Experimental Procedures’, Table 1;
Figure 2) to facilitate easier description and linkage
to MGEs.
Pseudoxanthomonas Sp. SE1
A GI of 123,035 bp overlapping the metal resistance
regions of D-J that we have designated GIPse
udoxSE1-1 (Figure 4A) was identified. GIPseud
oxSE1-1 contains 38-bp IRs, which are abutted by 5-bp
DRs and genes encoding a tyrosine recombinase and
a Tn3 family transposase at one end. First identified in
Stenotrophomonas maltophilia (Crossman et al., 2008),
the GI contains ISStma11, an IS of the ISL3 family con-
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COMPLEX GENOMIC REGIONS OF METAL RESISTANCE ASSOCIATED WITH NOVEL MGES 7

F I G U R E 3 Frequency of metal resistance genes on the genomes of Sphingobium sp. SA2, Sphingopyxis sp. SE2 and Pseudoxanthomonas
sp. SE1. A total of 101, 102 and 47 copies of metal resistance genes were identified across the genomic materials of Sphingobium sp SA2,
Sphingopyxis sp SE2 and Pseudoxanthonas sp SE1 respectively, conferring resistance to Hg, Cd, Pb, Cu and Zn.

taining merAPTR passenger genes (Figure 4A). Within
GIPseudoxSE1-1, a 20,650-bp Tn3 family transposon
designated Tn-PseudoxSE1-1 contains a mer operon
(Figure 4A) and is responsible for the region I (Table 1;
Figure 2). Tn-PseudoxSE1-1 is inserted in czcA of GIP-
seudoxSE1 and is abutted by 7-bp DRs. A GI in Steno-
trophomonas acidaminiphila ZAC14D2_NAIMI4_2
(Vinuesa & Ochoa-Sa nchez, 2015) has regions identi-
cal to GIPseudoxSE1 but lacks Tn-PseudoxSE1-1 and,
the cadA and czcCBAD genes of region D (Table 1;
Figure 2). Tn-PseudoxSE1-1 is duplicated on another
part of the chromosome (Table 1; Figure 2) and is
responsible for region C (Table 1). In addition to
ISStma11, an IS of the IS21 family designated ISP-
xasp4 contains passenger merRACPTR genes and
ISPxasp3 of the IS5 family (Figure 4B) and is responsi-
ble for region B (Table 1). The resistance genes in
regions A, K and L (Table 1; Figure 2) are not part of
any obvious mobile elements although, acr3 and arsH
of region A were absent from the closely related
genomes of Pseudoxanthomonas japonensis strains
TUM 20250 and DSM 17109 indicating either loss from
these strains or possible lateral acquisition in SE1
(Table S2).
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8 MAHBUB ET AL.

TABLE 1 List of metal resistance regions and their positions in replicon/contigs.

Region Position Metal resistance genes Metal resistance Mobile element Contig
Sphingobium sp. SA2; accession No.—SAMN31866039
Chromosome 1
A 624,316– merT, merBBBBTPAB Mercury ICESphspSA2-1 1
635,634
B 1,173,276– arsCC, acr3, arsH Arsenic – 1
1,173,797
C 1,406,034– czcCBA, czcD, cadA, czcD Cadmium/zinc/cobalt/lead GISphspSA2-1 1
1,420,104
D 1,438,005– cnrCBA Cadmium/zinc/cobalt/lead GISphspSA2-1 1
1,443,657
E 2,736,965– czcABC Cadmium/zinc/cobalt/lead – 1
2,742,530
Chromosome 2
A 78,409– cusFAB, copBA Copper 2
91,641
B 269,099– copD, pcoC, copA Copper 2
273,852
C 352,252– czcCBA, czcD, cadA Cadmium/zinc/cobalt/lead 2
363,126
D 374,357– nccX, copABB, copCD, Nickel/cadmium/zinc/cobalt/ Tn-SphspSA2-1 2
409,227 copA, actP, czcABC, lead/copper/cobalt/
copBA cadmium, copper
E 436,593– copAB Copper Tn-SphspSA2-1 2
439,282
F 452,498– copBA, nccX, copCD, silP Copper/nickel/cobalt/ Tn-SphspSA2-1 2
461,609 cadmium/silver
p172
A 7975–13,716 czcABC Cadmium/zinc/cobalt/lead – 4
B 63,355– merTPFAB Mercury – 4
66,223
C 71,067– merTPA Mercury Tn-SphspSA2-5 4
73,261
D 84,296– merT, copA. merBTPCTA, Mercury, copper, cobalt, – 4
105,103 copA, corC, chrA, chrA, chromium
copBA
E 134,692– cadI, arsC, acr3, arsB Cadmium, arsenic Abutted by overlapping DRs of 4
139,370 317-bp and 138-bp
F 139,919– copACA Copper –
141,459
G 151,887– merAB Mercury – 4
153,997
p58
A 24,717– copDCBA Copper – 6
33,264
Pseudoxanthomonas sp.SE1; accession No.—SAMN31866041
Chromosome
A 283,276– arsC, acr3, arsH Arsenic – 1
286,992
B 374,936– merACPT Mercury ISPxasp4 1
378,033
C 2,036,984– merEACPT Mercury Tn-PseudoxSE1-1 1
2,040,646
D 2,108,034– cadA, czcCBAD Cadmium/zinc/cobalt/lead GIPseudoxSE1-1 1
2,118,131
E 2,132,231– copBBA, actP Copper GIPseudoxSE1-1 1
2,147,792
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COMPLEX GENOMIC REGIONS OF METAL RESISTANCE ASSOCIATED WITH NOVEL MGES 9

TABLE 1 (Continued)

Region Position Metal resistance genes Metal resistance Mobile element Contig
a
F 2,154,314– merTPA Mercury ISStma11 > GIPseudoxSE1-1 1
2,156,661
G 2,160,247– cusBA Copper GIPseudoxSE1-1 1
2,164,898
H 2,182,445– silP, copBA Silver, copper GIPseudoxSE1-1 1
2,189,522
I 2,205,289– merTPCA Mercury Tn-PseudoxSE1-1 > 1
2,208,330 GIPseudoxSE1-1
J 2,217,542– czcABCD Cadmium/zinc/cobalt/lead GIPseudoxSE1-1 1
2,222,664
K 2,232,592– copAB, actP Copper – 1
2,238,711
L 2,384,962– czcABC Cadmium/zinc/cobalt/lead – 1
2,390,706
Sphingopyxis sp. SE2; accession No.—SAMN31866040
Chromosome
A 3,042,297– copBBA, nccX, pcoC, copD, Copper, nickel/cobalt/ Putative ICE 1
3,052,273 silP cadmium
B 3,060,230– cusBAF Copper Putative ICE 1
3,065,433
C 3,105,665– cadA, cnrABC, czcCBA, zitB, Cadmium/zinc/cobalt/lead, Putative ICE 1
3,124,413 czcD nickel
D 3,155,746– czcDCBA, actP Cadmium/zinc/cobalt/lead, Putative ICE 1
3,166,868 copper
E 3,208,311– copDCBA, nccX Copper, nickel/cobalt/ Putative ICE 1
3,217,421 cadmium
F 1,233,801– merAPT Mercury GISphpxspSE2-1 1
1,236,453
G 786,623– czcAABC Cadmium/zinc/cobalt/lead – 1
792,379
H 2,594,598– copDC, nccX, copAB Copper, nickel/cobalt/lead – 1
2,600,379
p231
A 1585,86– merACTPB Mercury – 3
166,198
B 185,963– merTBP Mercury – 3
192,396
C 194,723– merBAFT Mercury Tn-SphpxspSE2-1 3
197,661
D 217,059– merCPTPBATP Mercury – 3
227,497
Contig 2 (putative plasmid)
A 31,274– merTPAB, merTPA Mercury Tn-SphpxspSE2-5 2
42,970
B 50,811– merBTPF Mercury Tn-SphpxspSE2-5 2
53,061
C 58,534– merAPT, chrAB Mercury, chromium Tn-SphpxspSE2-6 > Tn- 2
60,727 SphpxspSE2-5
D 85,702– cnrA, czcBC Cadmium/zinc/cobalt – 2
91,406
E 286,696– merTPA Mercury Tn-SphpxspSE2-8 2
288,889
F 294,967– cadA, zitB, cnrA, czcBC, Cadmium/zinc/cobalt/lead/ – 2
309,679 cusFAB copper
G 321,523– nccX, copABCD Nickel/cadmium/cobalt – 2
327,078
a
ISStma11 > GIPseudoxSE1-1 indicates ISStma11 is inserted within GIPseudoxSE1.
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10 MAHBUB ET AL.

F I G U R E 4 Schematic representation of GIPseudoxSE1-1 containing ISStma11 and Tn-PsuedoxSE1-1 (A) and ISPxasp4 (B) on the
chromosome of Pseudoxanthomonas sp. SE1. All ORFS and their orientation are shown as block arrows with names given only for relevant
ORFs. Relevant genetic contexts are provided with a number in square brackets showing the amount of DNA sequence absent from the
drawing. Transposons and IS elements abutted by DR indicate acquisition by insertion and are provided in dashed boxes with the point of
insertion shown by a dashed line. Other IS elements are labelled with a line containing solid ends. IRs are shown as flags. Hg, Cd/Zn/Co/Pb, Cu
resistance ORFs are shaded red, blue and orange, respectively. Transposases and integrase ORFs are shaded in grey stripes. Bar = 0.5 Kb.

Sphingobium sp. SA2
Chromosome 1
Region A containing Hg resistance genes (Table 1;
Figure 2) is on a likely ICE of 75,494 bp due to the pres-
ence of genes involved in conjugation and is designated
ICESphspSA2-1 (Figure 5A). The ICE contains an inte-
grase gene encoding a tyrosine-type recombinase at
one end and is abutted by 16-bp DRs but no IRs were
observed. This GI is absent in one of SA2’s closest rela-
tives Sphingobium sp. RAC03 (NZ_CP016456).
Regions C and D containing Cd/Zn/Co/Pb resistance
genes (Table 1; Figure 2) are present on a 64,128-bp
GI inserted near a tRNA and designated GISphspSA2-1
(Figure 5B). Like ICESphspSA2-1, this GI is absent in
Sphingobium sp. RAC03 (NZ_CP016456). It is abutted
by 7-bp DRs and contains 26-bp IRs. GISphspSA2-1
contains a mosaic of diverse IS elements. Additionally,
multiple genes encoding proteins with predicted
transposition functions (tniA, tniB, tnpA, tnsA, tnpB) not
associated with IS elements were identified and may
have a role in GI mobility. The As resistance region B
and Cd/Zn/Co/Pb resistance region E on chromosome
1 (CH1) (Table 1; Figure 2) were not part of any obvious
mobile elements and are present in closely related
genomes (Table S2).
Chromosome 2
Interrogation of the sequence found it to be heavily
impacted by diverse mobile DNA including 23 different
IS elements from the IS3, IS5, IS6, IS21, IS30, IS66,
IS91, IS110 and IS256 families (Table 2). In many
instances, composite elements consisting of IS ele-
ments within IS elements were identified with one such
composite consisting of ISSphsp1 inserted within
ISSphsp11 inserted within ISSphsp21 inserted
within ISSphsp2 (Table 2). A RIT disrupted by
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COMPLEX GENOMIC REGIONS OF METAL RESISTANCE ASSOCIATED WITH NOVEL MGES 11

F I G U R E 5 Schematic representation of ICESphspSA2-1 (A) and GISphspSA2-1 (B) on chromosome 1, Tn-SphspSA2-1 (C) on
chromosome 2 and Tn-SphspSA2-5 (D) on p172 of Sphingomonas sp. SA2. All ORFS and their orientation are shown as block arrows with
names given only for relevant ORFs. Relevant genetic contexts are provided with a number in square brackets showing the amount of DNA
sequence absent from the drawing. Transposons and IS elements with evidence of a clean insertion are shown in dashed boxes with DRs
provided. Other IS elements and transposons are labelled with a line containing solid ends. IRs are shown as flags and Tn3 family transposons
are numbered to help identify their boundaries. Hg, Cd/Zn/Co/Pb, Cu and Cr resistance ORFs are shaded red, blue, orange and turquoise,
respectively. Transposases and integrases are shaded in grey stripes and conjugative ORFs as grey curved horizontal stripes. The green bfrD
ORFs in (C) share 95.6% identity. Bar = 0.5 Kb.

ISSphsp1 was identified as well as two putatively
mobile recombinases designated RI-Sphsp1 and RI-
Sphsp2. RI-Sphsp1 was found in isolation and inserted
in two IS elements of the IS21 family (ISSphsp7 and
ISSphsp9; Table 2). An 8675-bp sequence of
ISSphsp11 linked to 5S, 23S and 16S rRNA and three
tRNA genes in two genetic locations (Figure S1) is of
interest due to the common use of 16S rRNA as a taxo-
nomic marker. In one location (204,548–213,222; Loca-
tion 1), 8-bp DRs are observed abutting ISSphsp11
indicating a clean insertion near the RNA genes. In the
other location (606,968–615,642; Location 2), no DRs
are observed abutting ISSphsp11; however, the
ISSphsp11-RNA regions are identical indicating that
ISSphsp11 may have mobilized these rRNA and tRNA
as passenger genes. The rRNA sequences are identi-
cal to the lone 5S, 23S and 16S rRNA sequences pre-
sent on CH1.
Regions D–F (Table 1; Figure 2) are all present on a
large 144,553-bp Tn3 family transposon containing
34-bp IRs and designated Tn-SphspSA2-1 (Figure 5C).
Internal to Tn-SphspSA2-1 is a duplicate reverse IR pro-
ducing a 4257-bp Tn3 family transposon designated Tn-
SphspSA2-2 and contains genes encoding a Tn3 family
transposase, a resolvase and a GNAT family acetyl-
transferase. Tn-SphspSA2-2 is present on p58 abutted
by 6-bp DRs and on contig 2 of Sphingopyxis sp SE2
(99.9% identity over 100% coverage) indicating mobility
across species. Interestingly, a further two Tn3 family
transposons are contained within Tn-SphspSA2-1 desig-
nated Tn-SphspSA2-3 and Tn-SphspSA2-4 (Figure 5C).
Tn-SphspSA2-3 contains 33-bp IRs and a Tn3 family
transposase gene interrupted by ISSphsp12 and a resol-
vase gene. Tn-SphspSA2-4 contains 34-bp IRs and
genes encoding TonB-dependent receptors—no genes
encoding transposases or resolvases are present
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12 MAHBUB ET AL.

TABLE 2 Small insertion sequences on chromosome 2 of Sphingobium sp. SA2.

IS Insertion
IS name family Frequency position Relevant information
Insertion sequence elements
ISSphsp1 IS3 3 57,864 Part of an
ISSphsp1 > ISSphsp11 > ISSphsp21 > ISSphsp2
composite elementa
292,173 Part of an RIT_ Sphsp1 > ISSphsp1 composite
element
409,239 In isolation
ISSphsp2 2 55,718 Part of an
ISSphsp1 > ISSphsp11 > ISSphsp21 > ISSphsp2
composite elementa
91,652 In isolation
ISSphsp3 1 72,176 In isolation
ISSphsp22 1 28,809 In isolation
1 76,139 In isolation
ISSphsp5 1 467,029 Part of an ISSphsp18 > ISSphsp5 composite element
ISSphsp21 IS5 1 55,764 Part of an
ISSphsp1 > ISSphsp11 > ISSphsp21 > ISSphsp2
composite element
IS6100 IS6 4 162,674 In isolation
273,757 In isolation
288,271 In isolation
517,143 In isolation
ISSp5 IS21 2 307,890 In isolation
502,409 In isolation but is a partial element
ISSphsp7 5 129,760 In isolation
616,038 In isolation
654,321 Part of an RI_Sphsp1 > ISSphsp7 composite element
724,417 In isolation
1,106,057 Part of an RI_Sphsp1 > ISSphsp7 composite element
ISSphsp8 2 426,372 In isolation
993,508 In isolation
ISSphsp9 1 63,430 Part of an RI_Sphsp2 > ISSphsp9 composite element
ISSphsp10 IS30 3 74,959 In isolation
106,321 In isolation
809,833 In isolation
ISSphsp11 IS66 4 56,113 Part of an
ISSphsp1 > ISSphsp11 > ISSphsp21 > ISSphsp2
composite element
204,548 In isolation but likely mobile with 5S, 23S and 16S
rRNA cargo
282,532 In isolation
612,817 In isolation but likely mobile with 5S, 23S and 16S
rRNA cargo
ISSphsp12 2 305,669 In isolation
341,039 In isolation
ISSphsp13 1 480,084 In isolation
ISSphsp14 2 689,425 In isolation
1,111,709 In isolation
ISSphsp15 IS91 1 293,573 Part of an ISSphsp16 > ISSphsp15 composite
element
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COMPLEX GENOMIC REGIONS OF METAL RESISTANCE ASSOCIATED WITH NOVEL MGES 13

TABLE 2 (Continued)

IS Insertion
IS name family Frequency position Relevant information
ISSphsp16 IS110 1 294,797 Part of an ISSphsp16 > ISSphsp15 composite
element
ISSphsp17 2 117,744 In isolation
746,955 Part of an ISSphsp17 > ISSphsp20 composite
element
ISSphsp18 2 10,738 In isolation
467,014 Part of an ISSphsp18 > ISSphsp5 composite element
ISSphsp23 1 132,476 In isolation
ISSphsp20 IS256 3 30,813 In isolation
116,480 Part of an ISSphsp17 > ISSphsp20 composite
element
746,791 Part of an ISSphsp17 > ISSphsp20 composite
element
Putative recombinase insertion elements
RI-Sphsp1 (serine – 4 63,841 Part of an RI_Sphsp1 > ISSphsp9 composite element
recombinase) 498,168 In isolation
654,593 Part of an RI_Sphsp1 > ISSphsp7 composite element
1,106,329 Part of an RI_Sphsp1 > ISSphsp7 composite element
RI-Sphsp2 (tyrosine – 1 174,443 In isolation
recombinase)
Recombinase in trio element
RIT_Sphsp1 – 1 289,984 Part of an ISSphsp1 > RIT_ Sphsp1 composite
element
a
ISSphsp1 > ISSphsp11 > ISSphsp21 > ISSphsp2 indicates a composite element consisting of ISSphsp1 inserted within ISSphsp11 inserted within ISSphsp21
inserted within ISSphsp2.

(Figure 5C) and would require in trans transposition
activity of another Tn3 family transposon to mobilize. Tn-
SphspSA2-1 contains multiple ISs including ISSphsp1,
ISSphsp8, ISSphsp13, a composite ISSp
hsp5 > ISSphsp8 element, a partial ISSphsp6 and RI-
Sphsp1. Additionally, at one end of Tn-SphspSA2-1 is
bfrD encoding a TonB-dependent receptor and a partial
bfrD at the other end adjacent to a partial ISSphsp6. The
bfrD sequences share 95.6% identity over 637 nucleo-
tides and could homologously recombine excising the
multi-metal resistance region as a DNA circle.
The resistance genes in regions A, B and C
(Table 1; Figure 2) are not part of any obvious mobile
elements although, the cusFAB genes of region A are
absent in closely related strains of D43FB and RAC03
indicating possible lateral acquisition (Table S2).
any other mobile elements. In p172, a Tn3 family trans-
poson designated Tn-SphspSA2-5 was identified as
carrying the merAPTR of region C as passenger genes
(Figure 5D; Table S3). The arsC, acr3 and arsB
genes of region E did not appear to be part of a mobile
element but are abutted by overlapping DRs of 317 and
138 bp across a coding sequence encoding a hypothet-
ical protein. As a result of the DRs, this region could
excise as a circle through homologous recombination
and may be mobile (Figure S2). Similarly, while genes
encoding recombinases were present across the plas-
mid, the other metal resistance regions are not part of
any MGEs.
Sphingopyxis sp. SE2

Plasmids
Of the three plasmids, p172 and p58 contain metal
resistance regions (Table 1; Figure 2); however, ana-
lyses determined that all three plasmids are impacted
by Tn3 family transposons and ISs (Table S3).
Although Region A in p58 is closely surrounded by Tn3
family transposons and ISs, there was no evidence that
it had been mobilized into p58 by these elements or
Chromosome
Contig 1 is 4.6 Mb and is impacted by multiple GIs as
evidenced by five prophage integrases, three adjacent
to tRNAs. The metal resistance regions of A–E
(Table 1; Figure 2) are contained between two ISSph7
elements that are inverted relative to one another
(Figure 6A). The 526 Kb DNA region between these
two elements has likely inverted through homologous
recombination such that when inverted, 8-bp DRs of
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14 MAHBUB ET AL.

‘atcgcgtc’ for ISSph7 at the right-hand end would be
evident (Figure 6A). The region is complex with an
ISSph8, a group II intron, fragments of other IS ele-
ments and three integrase (int) genes; however, the
presence of two regions of conjugative genes indicates
the presence of two ICE elements. One of these
regions is 99% identical to a region in Altererythrobac-
ter sp. B11 (shaded box in Figure 6A) indicates mobility
and contains an integrase (int) gene and czcA. The
ends of the other putative ICE could not be determined
but its absence in the genomes of closely related
Sphingopyxis genomes supports lateral acquisition. On
contig 2 is a GI inserted adjacent to a tRNA and abutted
by 15-bp DRs (Figure 6B) containing the Hg resistance
operon of region F and designated GISphpxspSE2-1.
The czcAABC genes of contig 3 (region G) and copDC,
nccX and copAB of contig 8 (region H) are not linked to
any obvious MGEs.
Plasmids
Both p231 and contig 2 are heavily impacted by a vari-
ety of integrative MGEs that include transposons, IS
elements, an RIT element and a group II intron
(Table S4). p231 contains a region of �22 Kb encoding
conjugative genes and therefore, likely conjugative. It is
impacted by four Tn3 family transposons linked to Hg
resistance genes (Figure 7A). Tn-SphpxspSE2-1
contains the Hg resistance genes of region C (Table 1;
Figure 7A). The Hg resistance genes of region D sit
between Tn-SphpxspSE2-1 and Tn-SphpxspSE2-2
although would unlikely make a composite transposon
given the difference in IR sequences. Interestingly,
325-bp DRs (Figure 7A) that encompass a part of tnpA
of Tn-SphpxspSE2-1 and a region downstream of Tn-
SphpxspSE2-2 are present indicating the capability of
the region between these repeats to excise as a circle
through homologous recombination and may partly
explain how this region was acquired. Regions A and B
do not appear to be part of integrative MGEs. Contig
2 contains multiple Tn3 family transposons. Tn-
SphpxspSE2-5 is a large 63.5 Kb transposon contain-
ing Hg and Cr resistance regions contained within
regions A–C (Table 1) with region C contained
within Tn-SphpxspSE2-6 (Figure 7B). While Tn-
SphpxspSE2-7 is contained with Tn-SphpxspSE2-5, it
does not carry any MRGs. Tn-SphpxspSE2-8 is an
additional Hg-resistant transposon responsible for
region E (Figure 7C; Table 1). Regions D, F and G are
not part of any obvious integrative MGEs.
DISCUSSION
Interrogation of genome sequences is useful in deter-
mining the phenotypic potential of bacteria and provid-
ing insights into the evolutionary processes driving

F I G U R E 6 Genetic context of putative ICE elements (A) and GISphpxspSE2-1 (B) on the chromosome of Sphingopyxis sp. SE2. All ORFS
and their orientation are shown as block arrows with names given only for relevant ORFs. Relevant genetic contexts are provided with a number
in square brackets showing the amount of DNA sequence absent from the drawing. ISSph8 shows evidence of a clean insertion and is given in a
dashed box with DRs provided. ISSph7 elements are labelled with a line containing solid ends with the 526 Kb region between them showing
evidence of inversion such that if restored, 8-bp DRs for ISSph7 on the right-hand end would be evident. IRs are shown as flags. Hg, Cd/Zn/Co/
Pb and Cu resistance ORFs are shaded red, blue and orange, respectively. Transposases and integrases are shaded in grey stripes and
conjugative ORFs as grey curved horizontal stripes. The blue-shaded region in (A) is 99% identical to a region in Altererythrobacter sp. B11.
Bar = 0.5 Kb.
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COMPLEX GENOMIC REGIONS OF METAL RESISTANCE ASSOCIATED WITH NOVEL MGES 15

F I G U R E 7 Genetic context of p231 (A) containing Tn-SphpxspSE2-1, Tn-SphpxspSE2-2, Tn-SphpxspSE2-3 and Tn-SphpxspSE2-4; the
genetic context of contig 2 with Tn-SphpxspSE2-5 containing Tn-SphpxspSE2-6 and Tn-SphpxspSE2-7 (B); and Tn-SphpxspSE2-8 (C). All
ORFS and their orientation are shown as block arrows with names given only for relevant ORFs. Relevant genetic contexts are provided with a
number in square brackets showing the amount of DNA sequence absent from the drawing. Transposons and IS elements with evidence of a
clean insertion are shown in dashed boxes with DRs provided. Other IS elements and transposons are labelled with a line containing solid ends.
IRs are shown as flags and for Tn3 family transposons are numbered to help identify their boundaries. Hg and Cr resistance ORFs are shaded
red and turquoise, respectively. Transposases and integrases are shaded in grey stripes. The blue-shaded region in (A) indicates IRs consisting
of ISSph13 and part of the IR from Tn-SphpxspSE2-3. Solid black triangles on their sides in (A) represent DRs of 325 bp. Bar = 0.5 Kb.

genome structure (Land et al., 2015). In this study, we
sequenced the genomes of three metal-resistant bacte-
ria from the same soil, two with broader and higher
levels of resistance (SA2 and SE2) than the other
(SE1). While the frequency of MRGs in the genomes
helped explain the observed metal resistance pheno-
types, other factors such as intrinsic resistance associ-
ated with cellular physiology and the timing or level of
transcription/translation also play a role. The higher
resistance of SA2 to Cd and Zn could be explained by
the higher number of the czcABC and cadA efflux pump
genes. Pb resistance is driven by diverse genes includ-
ing czcABC, cadA, zntA and copA involved in efflux
and, the metallothionine proteins encoded by smtA
which sequesters metals. Among these Pb-resistant
determinants, czcABC, cadA and copA genes are in
higher copy in SA2 relative to SE2 despite levels of Pb
resistance being identical between the two strains. This
might be explained by additional Pb resistance genes
in SE2 not present in SA2 including smtA and zntA.
With regard to Cu resistance, SA2 has higher levels of
the copA efflux pump gene and cueO encoding a multi-
Cu oxidase; however, it lacks pcoC encoding a Cu-
binding resistance protein that is homologous to CopC.
Additionally, SA2 contains Cu resistance genes on mul-
ticopy plasmids that might increase their transcription
levels including copBA and copACA on p172 and,
copDCBA on p58. In addition to cop genes, SA2 and
SE2 contain cusFAB genes that encode an efflux chan-
nel. In SE2, the copy number of cusFAB is double that
of SA2 with one present on the multi-copy putative
plasmid (contig 2) potentially explaining the higher Cu
EC50 value. SE2 has a slightly higher EC50 than SA2
for Hg (4.5 mg/L vs. 5.9 mg/L), which might be
explained by a higher frequency of mer transport genes
(merT, merP and merC) and the mercuric reductase
gene (merA). Both SA2 and SE2 hold eight and five
copies of merB, respectively, indicating capacity to
resist high levels of organomercurials.
Compared to SA2 and SE2, SE1 had the least
amount of MRGs explaining, at least in part, its metal
resistance profile. In particular, a lack of some Cu and
Pb resistance genes was noted as well as a lower fre-
quency of genes that provide resistance to Cd and

16
Zn. Additionally, its Hg resistance is 3–4-fold lower than
SA2 and SE2, respectively, this is despite SE1 having
four copies of merA compared to five copies in SA2
and eight copies in SE2 although a lower intrinsic resis-
tance and/or a reduced amount of merT and lack of
merF transport genes could explain this result. SE1
carries no copies of merB indicating an inability to resist
organomercurials. Unlike SA2 and SE2, SE1 lacks
plasmids so none of its MRGs would be present in mul-
ticopy which could also explain its lower metal
resistance.
The genomes of all three strains showed significant
diversity of MGEs indicating a need for genetic variation
in highly selective environments and that LGT is impor-
tant in generating diversity. Indeed, metals are known
to enhance LGT processes such as conjugation (Pu
et al., 2021; Zhang et al., 2018) and in support, we
observed evidence of MGE sharing of ISSphsp21,
ISSp5 and Tn-SphspSA2-2 between SA2 and SE2.
While studies of metal resistance genomes often report
a high frequency of genes encoding transposases or
integrases and/or genetic elements such as transpo-
sons and ISs linked to MRGs (Gillings, 2017), they
often fail to interrogate the DNA sequence to identify
mobile units. Here, we carefully analysed the genomes
identifying multiple novel MGEs including plasmids,
GIs, ICEs, IS and RIT elements, and Tn3 family trans-
posons with most intermingling to form complex com-
posite elements. GIs and ICEs were a feature in all
chromosomes and themselves, were heavily impacted
by a variety of MGEs such as transposons and IS ele-
ments. This is a common feature of GIs and ICEs and
facilitates their diversification (Dobrindt et al., 2004). In
SE1, GIPseudoxSE1-1 carried ISStma11 and Tn-
PseudoxSE1-1 harbouring Hg resistance genes and all
GIs and ICEs carried a variety of IS elements and other
MGEs that have likely played a role in their acquisition
of MRGs. The boundaries of GIPseudoxSE1-1,
GISphspSA2-1, ICESphspSA2-1 and GISphpxspSE2-1
were easily identified through genome comparisons
with close relatives and the presence of IRs and/or
DRs. However, the putative ICE elements in SE2 were
more complex and whilst exhibiting evidence of mobility
such as the presence of integrases, conjugative genes
and being located near tRNAs, the boundaries could
not be identified. Across all GIs/ICEs, Cd/Zn/Co/Pb,
Cu, Ag, Hg and Cr resistance genes were common
indicating their importance in metal resistance.
Beyond GIs and ICEs, Tn3 family transposons were
common across all genomes mostly linked with Hg
resistance, a common feature of Tn3 family transpo-
sons (Nicolas et al., 2015). With regard to Hg resis-
tance, Tn-PseudoxSE1-1 is duplicated and inserted
within GIPseudoxSE1-1 and in another location of the
SE1 chromosome increasing gene copy number and
presumably enhancing Hg resistance. Plasmids in SA2
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MAHBUB ET AL.
and SE2 carried multiple Hg resistance Tn3 family
transposons including Tn-SphspSA2-5 on p172 in SA2
and Tn-SphpxspSE2-1 on p231 and Tn-
SphpxspSE2-5, Tn-SphpxspSE2-6 and Tn-
SphpxspSE2-8 on the putative plasmid (contig 2) in
SE2. Beyond Hg resistance, Tn-SphspSA2-1 in SA2 is
inserted in chromosome 2 (CH2) and carries resistance
to Cu, Cd/Zn/Co/Pb, Ag and Cr. Additionally, a number
of other Tn3 family transposons not harbouring passen-
ger genes were observed and sometimes containing IS
elements. This intermingling of MGEs provides an
opportunity for their diversification and continued diver-
sification of the bacterial genomes.
IS elements are diverse and those that move in a
‘cut-and-paste’ fashion can mobilize DNA as compos-
ite transposons. Evidence of composite transposition
(i.e., the presence of DRs) was not observed; however,
IS elements bordering MRGs were common and indi-
cate that composite transposition may have occurred
and that IS-mediated deletion events may be responsi-
ble for the loss of DRs bordering composite transpo-
sons. Additionally, “cut-and-paste” IS elements
carrying Hg resistance passenger genes were identi-
fied in SE1 including ISStma11 previously reported in
S. maltophilia (Crossman et al., 2008) and ISPxasp4
identified in this study. How this cargo was acquired is
unclear; however, homologous recombination is a likely
possibility. Beyond composite transposition, duplicate
IS elements in DRs can recombine to excise interven-
ing DNA as a circle allowing it to integrate into another
location. Additionally, IS elements forming IRs can
invert the intervening region as observed in the chro-
mosome of SE2 between the ISSph7 sequences. IS
elements that use a rolling circle-type transposition
mechanism such as ISSphsp15 in SA2 can sometimes
acquire adjacent DNA by terminating at a surrogate
rather than its terminal end (Siguier et al., 2015) as has
been observed with antibiotic resistance genes
(Partridge Sally et al., 2018). In SA2, an rRNA region
linked to ISSphsp11 of the IS66 family appears to have
been duplicated and mobilized; however, given that lit-
tle is known about the transposition of this family
(Siguier et al., 2015), the mechanisms are unclear.
Homologous recombination is often an important mech-
anism in genome evolution but is hard to identify as
definitive DNA signatures are not always present. Nev-
ertheless, repeat sequences were observed abutting
MRGs raising the possibility that they could excise as
circles through homologous recombination and may
have even been acquired in this fashion. For example,
in SA2, the bfrD sequences in Tn-SphspSA2-1 on CH2
or the 325-bp DR sequences in p231 abutting the As
resistance region in p172.
Overall, this study has, in detail, highlighted the
importance of MGEs and more importantly their com-
plex interaction in the evolution of metal resistance in

COMPLEX GENOMIC REGIONS OF METAL RESISTANCE ASSOCIATED WITH NOVEL MGES
three soil bacteria. New MGEs have been described
including plasmids, GIs, ICEs, IS elements and trans-
posons, which will greatly assist in future research
describing metal resistance genomes and the evolution
of MGEs.
A UT H O R C ON T R IB UT IO NS
Khandaker Rayhan Mahbub: Conceptualization;
investigation; methodology; validation; formal analysis;
writing – review and editing; visualization;
writing – original draft; data curation; software. Caro-
line Chénard: Writing – review and editing; visualiza-
tion; formal analysis; methodology. Steven Batinovic:
Formal analysis; writing – review and editing. Steve
Petrovski: Methodology; software; writing – review and
editing; formal analysis; data curation; resources. Fed-
erico M. Lauro: Methodology; software; formal analy-
sis; writing – review and editing; data curation;
resources. Md Hafizur Rahman: Visualization;
writing – review and editing. Mallavarapu Megharaj:
Writing – review and editing; resources. Ashley
E. Franks: Writing – review and editing; funding acqui-
sition. Maurizio Labbate: Conceptualization; investiga-
tion; funding acquisition; writing – original draft;
methodology; validation; visualization; writing – review
and editing; formal analysis; project administration;
data curation; supervision; resources; software.
C ON F L I CT OF I NT E R E S T S T A T E M E NT
The authors declare no conflicts of interest.
D AT A AV AI L AB IL IT Y S T A T E ME NT
Genomes have been uploaded to the National Center
for Biotechnology Information (NCBI) database and
contain BioSample accessions SAMN31866039,
SAMN31866040 and SAMN31866041 for Sphingobium
sp SA2, Sphingopyxis sp SE2 and Pseudoxanthomo-
nas sp SE1 respectively, BioProject PRJNA905125:
https://www.ncbi.nlm.nih.gov/bioproject/905125
ORCID
Khandaker Rayhan Mahbub https://orcid.org/0000-
0001-7086-9965
Steve Petrovski https://orcid.org/0000-0002-6304-
6853
Federico M. Lauro https://orcid.org/0000-0002-8373-
1014
Ashley E. Franks https://orcid.org/0000-0003-1664-
6060
Maurizio Labbate https://orcid.org/0000-0003-1428-
3382
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S U P P O R T I N G I N FOR MA TIO N
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How to cite this article: Mahbub, K.R., Chénard,
C., Batinovic, S., Petrovski, S., Lauro, F.M.,
Rahman, M.H. et al. (2023) Complex interactions
between diverse mobile genetic elements drive
the evolution of metal-resistant bacterial
genomes. Environmental Microbiology, 1–19.
Available from: https://doi.org/10.1111/1462-
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